Vol. 43 No.

6 SCIENCE IN CHINA (Series B) December 2000

Catalytic reaction mechanism of L-lactate dehydrogenase:
an ab initio study
HOU Ruobing ( )
1, 2
, CHEN Zhida ( )
1, 3
, YI Xianghui ( )
2
, BI-
AN Jiang( )
1
& XU Guangxian( )
1, 3
1. State Key Laboratory of Rare Earth Materials Chemistry and Applications, Department of Chemistry, Peking Uni-
versity, Beijing 100871, China;
2. College of Chemistry and Life Science, Guangxi Normal University, Guilin 541004, China;
3. PKU-HKU Joint Laboratory on Rare Earth Materials and Bioinorganic Chemistry, Peking University, Beijing 100871,
China
Correspondence should be addressed to Chen Zhida (email: relab@pku.edu.cn)
Received August 1, 2000
Abstract Studies on the catalytic reaction mechanism of L-lactate dehydrogenase have been
carried out by using quantum chemical ab initio calculation at HF/6-31G
*
level. It is found that the
interconversion reaction of pyruvate to L-lactate is dominated by the hydride ion H

R
transfer, and the
transfers of the hydride ion H

R
and proton H
+
R
are a quasi-coupled process, in which the energy
barrier of the transition state is about 168.37 kJ/mol. It is shown that the reactant complex is 87.61
kJ/mol lower, in energy, than the product complex. The most striking features in our calculated
results are that pyridine ring of the model cofactor is a quasi-boat-like configuration in the transited
state, which differs from a planar conformation in some previous semiempirical quantum chemical
studies. On the other hand, the similarity in the structure and charge between the H
+
R
transfer proc-
ess and the hydrogen bonding with lower barrier indicates that the H
+
R
transfer process occurs by
means of an unusual manner. In addition, in the transition state the electrostatic interaction between
the substrate and the active site of LDH is quite strong and the polarization of the carbonyl in the
substrate is gradually enhanced accompanying the formation of the transition state. These calcu-
lated results are well in accord with the previous experimental studies, and indicate that the charge
on the hydride ion H

R
is only +0.13e in the transition state, which is in agreement with the reported
semiempirical quantum chemical calculations.
Keywords: L-lactate dehydrogenase, ab initio calculation, reaction mechanism, transition state, enzymatic catalytic reac-
tion.
It is well known that L-lactate dehydrogenase(LDH) plays an important function in both
higher and lower organisms. This enzyme catalyzes the interconversion between pyruvate and
lactate with nicotinamide adenine dinucleotide (NADH) as a cofactor. The reaction is expressed as
CH
3
(CO)COO

+ NADH + H
+
CH
3
CH(OH)COO

+ NAD
+
(1)
in which the transfers of both the hydride ion and the proton are involved. The key residues in the
active site and their roles in the reaction had been identified by previous experimental investiga-
tions
[1 7]
. On the basis of the X-ray structure analysis for the ternary dogfish LDH-NADH-
oxamate complex, where oxamate is the pyruvate-analogue inhibitor with the methyl group in
588 SCIENCE IN CHINA (Series B) Vol. 43
pyruvate replaced by an amino group, it is shown that Arg-171 binds and orients the substrate, and
His-195 is a proton donor (fig. 1). The roles of other residues are also important, for example,
Arg-109 is able to activate His-195 and to stabilize the transition state (TS), Asp-168 plays a role
in modulation of the pK
a
value of His-195, while Val-138 orients the cofactor s carboxyamide side
chain. In addition, it has been found experimentally that the pro-R hydrogen atom of NADH is
transferred in the enzymatic reaction (1)
[4]
. Furthermore, theoretical calculations
[8]
have shown that
the transfer of the pro-R hydrogen atom is preferred when the nicotinamide glycoside of the
NADH is bound oppositely with respect to amide group.
Fig. 1. Scheme of active site residues of L-lactate dehydrogenase, substrate lactate, and the cofactor NAD
+
. The direc-
tion of the electron transfer expressed by arrows is the reaction progress from lactate to pyruvate.
In recent years, computational chemistry has made a significant contribution to the mecha-
nism of the enzymatic reaction (1). Some theoretical studies had been reported by Wilkie
[9]
,
Andrs
[10 12]
, Gelpi
[13]
and Ranganathan
[14, 15]
. Their theoretical studies adopted semiempirical
quantum chemical calculations, such as AM1, PM3 and hybride QM/MM. In semiempirical
quantum chemical calculations, the studies with the model LDH-NADH-pyruvate show that there
is only one TS for the hydride ion (

R
H ) transfer in the reaction and the possible reaction mecha-
nism is hydride ion transfer following protonation of the carbonyl in pyruvate. However, a poten-
tial energy surface obtained by using QM/MM
[15]
indicates that there are two TS in the reaction,
corresponding respectively to the transfers of hydride ion (

R
H ) and proton (
+
P
H ), in which the

R
H transfer step is the rate-limiting, prior to the
+
P
H transfer. The QM/MM calculated results
differ distinctly from the previous semiempirical QM calculations
[11,12,14]
both on the reaction
mechanism and on the energy difference between the reactant and product complexes. The reac-
tion energy from the reactant complexes (pyruvate) to the product complexes (lactate) in
eq. (1) is 52.25 kJ/mol for QM/MM
[15]
, but 26.75 and 20.48 kJ/mol for PM3 and AM1
[12]
, re-
spectively. Moreover, the semiempirical calculations
[11,12,14,15]
reported the planar conformation of
the nicotinamide ring in the TS structures which differs from the individual cofactor model ob-
No. 6 CATALYTIC REACTION MECHANISM OF L-LACTATE DEHYDROGENASE 589
tained by ab initio calculation
[8]
and calculation of AM1 potential energy surface
[16]
. On the other
hand, it should be pointed out that the TS structure obtained from potential energy surface of
QM/MM is only constructed by the two reaction coordinates corresponding to H
R
and H
P
+
trans-
fers and the QM method used in the QM/MM calculations is still semiempirical AM1 method.
Due to the confusion and uncertainty on the reaction mechanism, it is necessary to further inspect
previous results at high level theoretically. It is difficult, however, to simulate explicitly the com-
plicated enzymatic reaction. In addition, to our knowledge, ab initio methods have not been used
to study this enzymatic reaction mechanism so far. In the present work the ab initio molecular
orbital theory at the HF/6-31G* level is used in our calculations with a greater model of the reac-
tion system in order to investigate the roles of key side residues at the active site of LDH and gain
an insight into mechanism of the reaction (1).
1 Calculated models
In our calculations the models for catalytic reaction mechanism of L-lactate dehydrogenase,
including five parts, are taken from the Protein Data Bank entry 1LDM
[17]
.
The Raman spectra study
[7]
found that the form of the substrate in enzymatic reaction (1) is
pyruvate, and the carboxylate moiety of pyruvate remains unprotonated at LDH s active site. Thus,
the unprotonated pyruvate is adopted as the model substrate in our calculations. Because no at-
tempt of the ab initio method has so far been made to treat such a great system, in the meantime,
the ab initio calculation is expensive, in our calculations the models of LDH active center and the
model cofactor are properly truncated so as to save computational time. The guanidine parts of
Arg-171 and Arg-109 are conserved and all replaced by the H
2
N-CO-NH
2
group. The imidazole
nucleus substituted by the hydrogen atom is positioned in the location of the His-195. It is inter-
esting to note that Adams
[5]
suggested that the main effect of the amide group on the coenzyme
probably should provide the pyridine ring with the appropriate redox potential by means of a
conjungated double bond, rather than an additional site of interaction with the enzyme. On the
other hand, Eventoff
[18]
indicated that the nitrogen atom of the carboxyamide in the NADH can
interact with Ser-163 residue (omitted in fig. 1). Moreover, a hydrogen bonding interaction be-
tween the nitrogen atom of the carboxyamide in the NADH and the Val-138 residue can be found
in the Protein Data Bank entry 1LDM
[17]
. In addition, Price
[19]
indicated that there is a very im-
portant hydrogen-bonding interaction between the carbonyl oxygen of the side chain carboxyam-
ide on the NADH and the side chain of Lys-250 (omitted in fig. 1), which prefers to transfer the

R
H . It seems that the interaction between the carboxyamide group of the cofactor and the substrate
is not important, and the carboxyamide group affects little the substrate in the enzymatic reaction.
Therefore, we replace the carboxyamide moiety with a Cl atom and take 1,4-dihydro-3-Cl-
pyridine as an NADH model. The initial geometric coordinates for a complete super-molecule
composed of 48 atoms are extracted from the X-ray crystallographic structure anlysis
[17]
. Num-
bering of this system is depicted in fig. 2.
590 SCIENCE IN CHINA (Series B) Vol. 43
2 Computational method and details
All geometry optimizations have been
carried out by using the 4-31G basis set with
Gaussian 94 program
[20]
. Unlike the study on
other supermolecules
[15]
, where the attempt to
use the hill-climbing algorithm to locate the TS
geometry from reactant and product complexes
failed, thus we obtained successfully the TS
using Berny s
[21]
analytical gradient optimization
routines. Furthermore, it is shown in our calcu-
lations that the

R
H transfer is the rate-limiting
step in the enzymatic reaction and the TS just
has only one imaginary frequency. The energy
at the HF/6-31G* level is calculated on the basis
of the optimized geometries at the HF/4-31G.
The calculations on the TS are carried out in the following three steps. In the first step, the
geometry optimizations of the models NADH, NAD
+
, His-195, His
+
-195, and H
2
N-CO-NH
2
(Arg-
109 and Arg-171), and substrate pyruvate and lactate were performed respectively. In the second
step, according to the position of each part in the X-ray crystallographic structure analysis
[17]
for
our calculated models, we built up the start geometries of the models for the reactant and product
complexes by taking together NADH, His
+
-195, Arg-109, Arg-171 and the substrate pyruvate for
the reactant complex models with their individual optimized geometries in the first step, and also
together NAD
+
, His-195, Arg-109, Arg-171 and the substrate lactate for the product complex
models with their individual optimized geometries in the first step by using Cerius 2 modeling
program
[22]
. Then, the geometry optimizations of the reactant and product complex models have
been performed from partial groups to the whole complex, step by step. The third step is the loca-
tion of the TS between the reactant and product complexes, and verification of the TS geometry.
All calculations were carried out on the supercomputer, SGI Power Challenge R10000, at the
Network Information Center of Chinese Academy of Sciences.
3 Results and discussion
3.1 Transition state structure and the reaction path following
The TS obtained is a complex state, in which the transfers of

R
H and
+
P
H are quasi-coupled
at some extent, but the

R
H transfer precedes the
+
P
H transfer. The calculated structural parame-
ters r
1
, r
2
, r
3
, r
4
, and (fig. 2) of the TS, as well as the reactant and product complexes are given
in table 1.
Fig. 2. Numbering of the model supermolecular system and
the intermolecular structural parameters, r
1
, r
2
, r
3
, r
4
, and
in the two-dimensional view of the transition state. The dash
lines represent the intermolecular hydrogen bonding.
No. 6 CATALYTIC REACTION MECHANISM OF L-LACTATE DEHYDROGENASE 591
Table 1 Structural parameters r
1
, r
2
, r
3
, r
4
(nm), () and () for the TS, RC and PC
a)
r
1
r
2
r
3
r
4

RC 0.245 8 0.107 5 0.101 7 0.247 8 135.0 138.0
TS 0.120 2 0.145 9 0.104 5 0.162 0 164.1 168.6
PC 0.107 0 0.241 9 0.189 0 0.097 3 114.8 158.7
a) RC: Reactant complex; PC: product complex.
The structural parameters r
2
and r
3
for TS, reactant complex and product complex in table 1
indicate that in the TS the
+
P
H starts to transfer while the

R
H is transferring about halfway. This
calculated result contrasts with the previous semiempirical quantum chemical calculated re-
sults
[11,12,14]
, in which one TS was found to be involved only in

R
H transfer state, as well as with
the hybride QM/MM calculation
[15]
. The later indicates two TS,

R
H transferred states and

R
H
transferred state respectively.
In order to verify that this TS structure obtained in our calculations actually corresponds to a
first-order saddle point and further this saddle point smoothly connects the potential energy mini-
ma corresponding to the reactant and product complexes, the reaction path following to the reac-
tant complex has been performed. It is shown that the TS is, indeed, reasonable because both the
final coordinates and the final energy in the reaction path following are very close to the reactant
complex. The results are listed in table 2. The vibration frequency calculated for the TS found that
the TS factually has only one imaginary frequency, 1 011.38 cm
1
. The displacement vectors of
this imaginary vibration mode in TS are shown in fig. 3.
Table 2 The reaction coordinates (S) and selected internal coordinates in the reaction path following
a)
S C2-C1 H
P
-N1 H
R
-C4 H
S
-C4 E
TS 0.153 4 0.104 5 0.145 9 0.107 2 102.7
0.147 0.153 6 0.103 9 0.139 7 0.107 3 103.2
0.297 0.153 8 0.103 4 0.133 5 0.107 3 103.6
0.447 0.153 9 0.102 9 0.127 2 0.107 4 104.0
0.567 0.154 0 0.102 5 0.121 3 0.107 5 104.3
0.747 0.154 4 0.102 2 0.116 2 0.107 6 104.6
0.896 0.154 2 0.101 8 0.112 3 0.107 6 105.0
1.045 0.154 2 0.101 6 0.110 0 0.107 7 105.4
1.195 0.154 1 0.101 5 0.108 8 0.107 7 105.8
1.345 0.154 0 0.101 4 0.108 1 0.107 8 106.4
1.494 0.153 8 0.101 3 0.107 6 0.107 9 106.9
1.644 0.153 4 0.101 2 0.107 3 0.107 9 107.4
1.794 0.153 4 0.101 1 0.107 1 0.108 0 108.0
1.944 0.153 2 0.101 1 0.106 9 0.108 0 108.5
2.009 0.153 1 0.101 1 0.106 8 0.108 1 108.9 1 717.720
RC 0.152 5 0.100 9 0.107 1 0.108 5 110.4 1 717.725
a) TS: transition state; RC: reactant complex; C2-C1, H
P
-N1, H
R
-C4 and H
S
-C4 are distances (nm) between C2 atom and
C1 atom on the substrate, H
P
atom and N1 atom on the residue His-195, H
R
atom and C4 atom in the cofactor, H
S
atom and C4
atom on the cofactor, respectively; is the angle H
R
-C4-C5 on the cofactor (fig. 2), in (); the total energy E is in a.u.
592 SCIENCE IN CHINA (Series B) Vol. 43
Fig. 3. The displacement vectors of the imaginary vibration mode in the TS. (a) The arrows pointing to the reactant com-
plex; (b) the arrows pointing to the product complex.
3.2 Reaction mechanism
In the enzymatic reaction (1), as mentioned above, the H
R
transfer precedes the
+
P
H transfer
and the

R
H and
+
P
H transfers are quasi-coupled. Because the carbonyl C== O bond of the sub-
strate in the reaction is strongly polarized by the guanidino group of Arg-109 and imidazole ring
of Hia-195, there is more positive charge on the C2 atom in the substrate, which leads the

R
H to
transfer forward the substrate preceding the
+
P
H . Why is not the
+
P
H transfer to O3 atom of the
substrate prior to the

R
H under the circumstance that there is more negative charge on O3 atom
of the substrate? We suggest that the quasi-boat-like conformation of the cofactor should be favor-
able to the

R
H transfer.
The whole reaction process is in fact quite complicated from the displacement vectors of the
imaginary vibration mode in which the
+
P
H and

R
H transfers are quasi-coupled in a sense. But it
should be pointed out that the
+
P
H and

R
H transfers are neither an interrelated transfer nor inde-
pendent transfer process each other. Thus, we suggest the reaction process as follows.
After the pyruvate substrate entering into the active site of LDH in the binary complex LDH-
NADH, the carbonyl C == O bond in the substrate is significantly polarized by the Arg-109 and
His-195 through the hydrogen bonding interaction. It is interesting to note that in such a circum-
stance there is a possibility of the transfer of either

R
H or
+
P
H to the substrate due to the dis-
tance of 0.245 8 nm between the

R
H and C2 atom in the substrate, being about the same as the
distance of 0.247 8 nm between the H
P
+
and O3 atom in the substrate. However, at this time, the
conformation of the cofactor changes suddenly from a planar one to the quasi-boat-like one which
shortens effectively the distance between

R
H and C2 in the substrate. It is the most important
No. 6 CATALYTIC REACTION MECHANISM OF L-LACTATE DEHYDROGENASE 593
change in the cofactor conformation that causes the

R
H transfer to occur first. It should be point-
ed out that
+
P
H transfer process has actually started to occur and has just progressed to a small
degree while the

R
H transfer process has progressed about a half of the way according to the
displacement vectors of the imaginary vibration mode in TS. Accompanying with the

R
H trans-
fer to the substrate, the residue His-195 also moves gradually to close to the substrate, and this
process is accelerated by the electrostatic interaction which results from more negative charge on
the O3 atom in the substrate following with

R
H transfer. It is evident that it is this change of
electrostatic interaction that triggers the transfer of
+
P
H in the His-195 to the O3 atom in the sub-
strate. It is necessary to point out again that though the

R
H transfer process has not yet been
completed, the
+
P
H starts to transfer, which is the reason why the

R
H and
+
P
H transfers are
recognized as a quasi-coupled process. The detailed
+
P
H transfer mechanism will be discussed
below.
3.3
+
P
H transfer mechanism
Attempts to obtain a TS, corresponding to the individual
+
P
H transfer from the imidazolium
ion to the deprotonated lactate substrate after the

R
H having been transferred to the substrate,
had been made, but the result is negative. This is in accordance with some previous semi-
empirical calculations
[11,12,14]
. It seems that there is not a TS of
+
P
H transfer. Some previous stud-
ies
[22]
also indicated that it is not true that all enzymatic reactions with the proton shift have their
TS. What is the reason leading to the disappearing of TS of the
+
P
H transfer and how does the
+
P
H transfer happen? These probably result from the formation of the hydrogen bonding
[23, 24]
with
a lower barrier between O3-H
P
-N1.
The
+
P
H transfer, in our opinion, occurs by means of the lower barrier hydrogen bonding
mentioned above. In the case of the lower barrier hydrogen bonding
[23, 24]
, in general, there is a
double potential well with a low barrier (about 8.36 kJ/mol) toward hydrogen transfer and the
hydrogen bonding in enzymatic reaction will consist of largely charged atoms. In most cases, the
distance between heteroatoms, such as NHO, is less than 0.265 nm, and as the NO distance
is compressed, the potential barrier between the wells will decrease in height, and further if the
distance is less than 0.255 nm, the hydrogen atom can freely move between the two wells. Be-
cause the structural and charge characteristics in the
+
P
H transfer step of the enzymatic reaction
are all in accordance with that of the lower barrier hydrogen bonding, it is reasonable to suggest
that the
+
P
H transfer occurs by means of the lower barrier hydrogen bonding. If the
+
P
H transfer
594 SCIENCE IN CHINA (Series B) Vol. 43
occurs really like what the above-mentioned, there should be, thus, a TS corresponding to the
+
P
H
transfer although its barrier is lower. But why does not we obtain the TS of
+
P
H transfer in our
calculations? First of all, the
+
P
H transfer, just as we have mentioned above, is a quasi-coupled
process with the

R
H transfer and once the

R
H is transferred in the TS, the lower barrier hydro-
gen bonding N1H
P
O3 noted above is formed. The next, after the

R
H closes further to the
C2 atom in the substrate, forming of the C2 H
R
bonding, the negative charge on the O3 atom of
the substrate would be more than that in TS, which results in the enhancing of the electrostatic
interaction between the O3 and
+
P
H atoms, with shortening of the distance between these two
atoms. On the other hand, as the

R
H transfers, the orbital hybride form of the C2 atom in the
substrate would be changed gradually from sp
2
to sp
3
, which would lead to a gradual change of the
C2 O3 bond from a double bond to a single bond with lengthening of the distance of C2 O3.
All these changes would cause the shortening of the bond length of the lower barrier hydrogen
bonding. It is important to recognize that the barrier of the hydrogen bonding will decrease so
much that the barrier disappears. It is this change in the potential surface that leads the TS of the
+
P
H transfer to vanishing in our calculations.
It should be noted that the present ab initio result shows that the LDH enzymatic reaction
mechanism, indeed, is a very complicated one, and the electronic structural characteristics on the

R
H and
+
P
H transfers, hence, need to be further studied so as to find out the reaction mechanism
more explicitly.
3.4 Energy of the reaction
The calculated active energy is 168.37 kJ/mol at the HF/6-31G* level for interconversion
from the pyruvate to the lactate, and our calculations also indicated that the reactant complex is
87.61 kJ/mol lower in energy than the product complex. This energy difference is greater than that
from all the semiempirical calculation results
[12,15]
. Due to the quasi-coupled transfer mechanism
of the

R
H and
+
P
H , it can be expected that the active barrier possibly contains a partial energy
required by
+
P
H transfer process. It should be noted that the energy of the reactant complex in the
QM/MM calculation, in which the QM method is AM1, is not lower, but 52.25 kJ/mol higher than
that of the product complex, and is different from our and the other calculation results (PM3 26.75
kJ/mol; AM1: 20.48 kJ/mol)
[11,12]
.
3.5 Ionic charge distribution during the reaction
Accompanying the preceding of the enzymatic reaction, much remarkable charge transfer
process happens. The net charges on partial atoms in the TS, reactant and product complexes are
given in table 3 on the basis of Mulliken population analysis. In the TS the hydrogen anion,
No. 6 CATALYTIC REACTION MECHANISM OF L-LACTATE DEHYDROGENASE 595

R
H , is not an anion in truth but a cation with +0.13e of the positive charge, which agrees with
that of the individual

R
H transfer step reported in the QM/MM calculation
[15]
, but is little more
than the other semiempirical results
[11,12]
.
Table 3 Net charges for substrate (SUB), hydride ion (H
R

),
+
P
H , H
S
, C2 and O3 in the substrate, C4 in the cofactor,
cofactor analogue (NIC), His-195 (HIS) species in the TS, the reactant and product complexes (RC and PC)
a)
RC
TS(H
R

)
PC
NIC 0.003 0.536 0.946
H
S
0.189 0.315 0.342
C4(NIC) 0.280 0.148 0.001
H
R

0.195 0.131 0.147

SUB 0.802 1.238 0.845
C2(SUB) 0.460 0.193 0.097
O3(SUB) 0.637 0.847 0.877
H
P
+
0.532 0.544 0.526
HIS 0.917 0.814 0.037
a) Atomic numbering in fig. 2.
It is interesting to note that the negative charges of ca. 0.10e are transferred to His-195 and
ca. 0.43e to substrate while the cofactor loses ca. 0.53e charge, accompanying the formation of
the TS. The charge transfer should be completed along the atom chain C4 H
R
C2 O3 H
P
N1 (C4
in the cofactor,

R
H , C2 and O3 in the substrate,
+
P
H and N1 in the His-195 residue) and this
charge transfer process is similar to the electron delocalization in a sense.
3.6 Structure changes of the substrate, LDH and cofactor in the reaction
In the enzymatic reaction (1), the change in partial structure of the substrate has taken place.
The distance between C2 and O3 (table 4) enlarges gradually from 0.121 8 nm to 0.143 6 nm,
which shows that the C== O double bond becomes gradually to be the CO si ngle bond and the
hybride form of the C2 atom changes gradually from sp
2
to sp
3
. The little change of the inversion
angle C1(O2,O1,C2) in the reaction process shows that the carboxylate moiety of the substrate is
Table 4 The selected geometric parameters for the reactant complexes (RC), TS and product complex (PC)
at HF/4-31G level
a)
RC TS PC
Inversion angles
C1(O2,O1,C2) 0.2 1.4 4.0
Dihedral angles
O1 C1 C2 O3 15.7 61.2 53.4
O1 C1 C2 C3 163.6 74.6 73.3
Distance
C2(sub) O3(sub) 0.121 8 0.133 6 0.143 6
C2(sub) C3(sub) 0.148 8 0.152 1 0.152 3
C4(cof) H
R
(cof) 0.107 5 0.145 9 0.241 9
H
R
(cof) C2(sub) 0.248 5 0.120 2 0.107 0
C4(cof) C2(sub) 0.333 3 0.263 6 0.303 8
N1(his) H
P
(his) 0.101 7 0.104 5 0.189 0
H
P
(his) O3(sub) 0.247 8 0.162 0 0.097 3
a) sub: substrate; cof: cofactor; his: His-195; distance in nm; angle in ().
596 SCIENCE IN CHINA (Series B) Vol. 43
really oriented by residue Arg-171 so that the reaction can proceed normally. This is in agreement
with the experimental results
[2, 7, 25]
. The changes of dihedral angles O1-C1-C2-O3 and O1-C1-
C2-C3 show that the substrate structure must be changed so that the reaction can take place nor-
mally. Indeed, the induced-fit interaction between the substrate and the residues of LDH occurs in
the enzymatic reaction (1).
In comparison to the reactant complex, it is found that except the atoms in Arg-109, the dis-
tances between atoms in the substrate and atoms of the residues in LDH are almost shortened in
TS. It is obvious that LDH controls the substrate more tightly in TS than that in the reactant com-
plex, and leads to the enzymatic reaction proceeding normally, according with some experimental
results
[5,19]
.
The most striking characteristic in the TS obtained by our calculation is that the configuration
of the pyridine part of the cofactor NADH is not planar but quasi-boat-like (fig. 4). On the pyri-
dine of the cofactor, the N1 atom leans to the substrate and the angle between the N1-C2-C6 plane
and the C2-C3-C6 plane is 2.6, and the angle between the N1-C2-C6 plane and the C2-C5-C6
plane is 3.7. The C4 atom on the pyridine also leans towards the substrate. It is shown in our
calculations that the angle between the C3-C4-C5 plane and the C2-C3-C5 plane is 13.1, and the
angle between the C3-C4-C5 plane and the C3-C5-C6 plane is 12.0. These calculated results are
in accordance with the results of individual model cofactor computed by ab initio
[8]
and AM1
calculations
[16]
. In our calculations, the energy demanded by the change in geometry configuration
for the cofactor is 37.79 kJ/mol. It can be expected that this energy can be partially compensated
by the decrease of the transition barrier in the TS. The

R
H transfer becomes easy after the

R
H
closes to the C2 atom in substrate pyruvate due to transformation of the configuration of the co-
factor to the quasi-boat-like one.
Fig. 4. Dihedral angles in the cofactor with the quasi-boat-like configuration in TS.
3.7 Carbonyl polarization in the substrate
It is found in our calculated results that there is a significant change in polarization of the
C2 O3 bond of the substrate in the reaction process. The dipole moment of the C2 O3 bond in
No. 6 CATALYTIC REACTION MECHANISM OF L-LACTATE DEHYDROGENASE 597
the substrate is calculated from the Mulliken population and the bond distances in the reactant
complex and TS at HF/4-31G level. The following formula is used in our calculations:
= q l, (2)
where is the dipole moment, q is the net charges (Mulliken population charge) on the C2 or O3
atom of the substrate and l is the distance between the C2 and O3 atoms in the substrate. The net
charges on the C2 and O3 atoms, the distance between the C2 and O3 atoms, and the dipole mo-
ment of the carbonyl in the substrate are listed in table 5.
Table 5 The dipole moments (Debye) of carbonyl, the C2 O3 distance (nm) and the net charges(e)
on C2 and O3 in the reactant complex (RC) and TS
Net charge
Dipole moment of carbonyl Distance of C2 O3
C2 O3
RC 6.441 0.121 8 +0.460 0.637
TS 6.669 0.133 6 +0.193 0.847
It is found that the dipole moment of the carbonyl in the substrate of reactant complex is
greater than that in TS, which shows that the dipole moment of the carbonyl in the substrate in-
creases really accompanying the formation of TS and it favors the
+
P
H transfer. This calculated
result agrees with the experimental results
[2, 7, 25]
.
4 Conclusions
The study on the reaction mechanism from pyruvate to lactate in the active site of the LDH
enzyme has been carried out by using quantum chemical ab initio method at HF/6-31G* level.
The optimized geometries in the ground state and TS had been obtained for the properly truncated
models. The calculated results can be summarized as follows:
(i) The transition state and the displacement vector analyses of its imaginary frequency show
that although the

R
H transfer happens prior to the H
P
+
transfer, both the transfers are not a two-
step process but a quasi-coupled one, namely, when the process of the

R
H transfer has not com-
pleted yet, the
+
P
H transfer process has begun to occur. In fact, there is not an individual
+
P
H
transfer step. Thus, the rate-limiting step is the

R
H transfer step. It has been verified in frequency
calculation of the transition state and reaction path analysis that our calculated results are reason-
able and acceptable. The only imaginary frequency of the transition state is 1 011.38 cm
1
.
(ii) The calculated active energy is 168.37 kJ/mol, which contains the partial energy required
for the H
P
+
transfer process on the basis of the cooperated transfer of

R
H and H
P
+
. The energy of
the reactant complex is 87.61 kJ/mol lower than that of the product complex.
(iii) In the TS the configuration of the pyridine part in the cofactor NADH is quasi-boat-like
one which is in favor of the

R
H transfer prior to the
+
P
H transfer. This result is in accordance
598 SCIENCE IN CHINA (Series B) Vol. 43
with that of the individual model NADH in ab initio study. The ab initio calculation at HF/4-31G
level shows that the change of the configuration of the model NADH requires the energy of 37.79
kJ/mol. The hydride anion,

R
H , is not an anion in truth but a cation with the positive charge of
+0.13e.
(iv) Accompanying the formation of TS, the LDH enhances its control to the substrate
through residues of the LDH in the active site, and the polarization effect of the carbonyl group
C2 O3 is furhter enhanced, which promotes the
+
P
H transfer. These calculated results are in
agreement with the experimental results.
(v) The similarity between the
+
P
H transfer process and the lower barrier hydrogen bonding
in the structure and charge distribution indicates that the
+
P
H transfer occurs by means of an un-
usual manner.
Acknowledgements We are grateful to the member of Chinese Academy of Sciences, Professor Li Lemin for informa-
tive discussion. This work was supported by the National Natural Science Foundation of China (Grant Nos. 29831010, 20023005
and 29863001) and State Key Project of Fundamental Research of China (Grant No. G1998061306).
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